Anti-tumor drug, medicament, composition, and use thereof

ABSTRACT

An active polypeptide comprising the amino acid sequence of SEQ ID NO:4, having an anti-tumour activity, and compositions and methods including the active polypeptide.

The present invention relates to the field of treatments for cancers.More specifically, the present invention relates to the treatment ofcancers by small polypeptides.

Cancer is a class of diseases or disorders characterized by uncontrolleddivision of cells and the ability of these cells to spread, either bydirect growth into adjacent tissue through invasion, or by implantationinto distant sites by metastasis (where cancer cells are transportedthrough the bloodstream or lymphatic system). Cancer may affect peopleat all ages, but risk tends to increase with age. It is one of theprincipal causes of death in developed countries.

There are many types of cancer. Severity of symptoms depends on the siteand character of the malignancy and whether there is metastasis. Oncediagnosed, cancer is usually treated with a combination of surgery,chemotherapy and radiotherapy. As research develops, treatments arebecoming more specific for the type of cancer pathology. Drugs thattarget specific cancers already exist for several cancers. If untreated,cancers may eventually cause illness and death, though this is notalways the case.

Current treatments target distinct properties of malignant cells, suchas for example evading apoptosis, unlimited growth potential(immortalization) due to overabundance of telomerase, self-sufficiencyof growth factors, insensitivity to anti-growth factors, increased celldivision rate, altered ability to differentiate, no ability for contactinhibition, ability to invade neighbouring tissues, ability to buildmetastases at distant sites, ability to promote blood vessel growth(angiogenesis).

Tumor angiogenesis is the proliferation of a network of blood vesselsthat penetrates into the tumor, supplying nutrients and oxygen andremoving waste products. Tumor angiogenesis actually starts withcancerous tumor cells releasing molecules that send signals tosurrounding normal host tissue. This signalling activates certain genesin the host tissue that, in turn, make proteins to encourage growth ofnew blood vessels. Solid tumors must stimulate the formation of newblood vessels in order to obtain the nutrients and oxygen necessary fortheir growth, thus providing a route by which the tumors can metastasizeto distant sites.

Experimental evidence has suggested that malignant tumors can induceangiogenesis through the elaboration of a variety of factors, such asacidic fibroblast growth factor (aFGF), basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF), platelet derivedgrowth factor (PDGF), transforming growth factor alpha (TGF-alpha),tumor necrosis growth factor alpha (TNF-alpha), and many others (Liottaet al., 1991, Cell 64: 327-336; Hanahan et al., Cell 86: 353-364).

Nowadays, plenty of chemotherapeutic molecules targeting angiogenesisare available on the market. Well known naturally occurring angiogenesisinhibitors are angiostatin, endostatin, interferons, platelet factor 4,prolactin 16 Kd fragment, thrombospondin, TIMP-1 (tissue inhibitor ofmetalloprotease-1), TIMP-2 and TIMP-3. These molecules can be used aschemotherapeutic treatments, as well as other drugs such as for examplecombrestatin A4, EMD 121974, TNP-470, squalamine, thalidomide,interferon-alpha, anti-VEGF, antibodies . . . . However, theirefficiency is never sufficient and alternative treatments are desirable.

There is therefore a need of alternative chemotherapeutic agents for thetreatment of tumors, having increased efficiency, being less invasive ortoxic, and resulting in an increased rate of recovery.

WO 03/080105, in the name of the Applicant, describes five genesinvolved in the regulation of angiogenesis. Amongst these genes, “gene168” (SEQ ID N^(o) 1 in this specification), which encodes “protein168A” (SEQ ID N^(o) 2 in this specification), has been described asimplied in the activation of angiogenesis. In particular, WO 03/080105discloses that protein 168A is expressed in endothelial cells stimulatedwith pro-angiogenic factors, such as for example TNF-α. WO 03/080105also describes that the expression, in human endothelial cells, of anantisens sequence of gene 168, i.e. the inhibition of the expression ofgene 168, inhibits the formation of capillary tubes.

In silico experiments further revealed that protein 168A, which isconstituted of 924 aminoacids, may have a single transmembrane domain,and five Immunoglobulin-like domains.

Going deeper in their researches, the inventors produced truncated formsof protein 168A, corresponding to various fragments of protein 168A.Amongst these fragments, 168A-T2 corresponds to a fragment of theextracellular domain of protein 168A, and is identified by SEQ ID NO:4in this specification (108 amino acids).

In a first experiment, the inventors found that protein 168A-T2 mayinhibit human endothelial cell proliferation in vitro in a dosedependent manner.

Then, in a second experiment, the inventors surprisingly found that168A-T2 may have a strong activity to inhibit capillary tube formationin vitro, in a dose dependent manner.

Other experiments conducted by the inventors suggested that 168A-T2 mayinduce the inhibition of the migration of endothelial cells in vitro, ina dose dependent manner.

The results of the dose-response study further revealed that the protein168A-T2 may inhibit in a dose-dependent manner the proliferation ofhuman endothelial cells, and this inhibition could reach more than 80%with a concentration of 3.1 μM. This tends to demonstrate that therecombinant protein may be a potent anti-angiogenic compound, at least600-fold more potent than the anti-VEGF mAb and/or VEGF receptor(KDR)-based identified peptides (Binetruy-Tournaire R et al.,Identification of a peptide blocking vascular endothelial growth factor(VEGF)-mediated angiogenesis, EMBO J. 2000; 19: 1525-1533).

Capillary tube formation, human endothelial cell proliferation and humanendothelial cell migration are three essential steps of angiogenesis.Consequently, the fact that 168A-T2 may inhibit in vitro capillary tubeformation, human endothelial cell proliferation and/or migration in adose-dependant manner, thus constituted a strong evidence of the potentanti-angiogenic activity of the truncated forms of protein 168A.

All these results were absolutely unexpected since native protein 168Ais preferably expressed in pro-angiogenic conditions, i.e. in thepresence of TNFα, and that the expression of an antisens of the gene168, i.e. the inhibition of gene 168, in human endothelial cellsinhibits the formation of capillary tubes. It was therefore reallysurprising that a truncated form of protein 168A, may haveanti-angiogenic activity.

Still surprisingly, the inventors found that protein 168A-T2 had astrong anti-tumor activity in vivo, and a strong synergistic activity incombination with other chemotherapeutic agent such as for examplecisplatin.

Inventors found that the test substance 168A-T2 was not toxic in Nudemice bearing tumors at different tested doses. Moreover, 168A-T2exhibited a strong statistically significant anti-tumoral activityagainst human tumors as early as two days after the beginning of thetreatment. This anti-tumoral activity was persistent during thetreatment period. This anti-tumoral effect of 168A-T2 represented arealistic therapeutic approach as a monotherapy. Its efficacy was alsostrongly potentiated when combined with the cytotoxic anticancer drugCDDP (Cisplatin), which suppressed tumor growth. Cisplatin alone, on theother hand, did not eradicate tumor growth.

The data strongly suggested that 168A-T2 may be of use either as aprimary anti-tumoral agent or as an add-on synergic therapy to primarycytotoxic agents for the treatment of cancers.

As mentioned above, it is now established that protein 168A is expressedin endothelial cells, and that its expression is enhanced whenangiogenesis is stimulated by pro-angiogenic factors such as TNFα, asdescribed in WO03/080105. Protein 168A is a transmembrane protein, andmight be implied in the transduction of a pro-angiogenic signal. Withoutwanting to be bound with a theory, Applicants suggest that the truncatedforms of 168A may play their role through a “soluble receptormechanism”: truncated forms of 168A may remain soluble on the surface ofthe cell and may be recognized by the ligand of the native 168A protein.As a result, there may be a competition in the recognition of the ligandbetween the soluble forms of 168A (the fragments of the invention) andthe native transmembrane protein, and consequently a decrease, in a dosedependent manner, of the transduction of the pro-angiogenic signal,therefore resulting in the inhibition of angiogenesis and then in thedecrease of tumour volume.

The invention thus relates, in a first aspect, to a nucleic acid havingthe nucleic acid sequence of SEQ ID NO:3, or having at least 50%,preferably 70%, more preferably 90% identity with the nucleic acidsequence of SEQ ID NO:3, or fragments thereof having at least 60contiguous nucleotides, or nucleic acid sequences having at least 50%,preferably 70%, more preferably 90% identity with the nucleic acidsequence of said fragments, provided that said nucleic acid is not SEQID NO:1, SEQ ID NO: 21, SEQ ID NO: 58 disclosed in US2005/106644 or SEQID NO: 23, said nucleic acid coding for a polypeptide or peptide havinganti-angiogenic and anti-tumour activity.

SEQ ID NO: 21 corresponds to the nucleic acid sequence SEQ ID NO: 87disclosed in WO02/081731.

SEQ ID NO: 23 corresponds to the nucleic acid sequence SEQ ID NO: 28disclosed in WO03/080105.

In a preferred embodiment, the invention relates to the nucleic acidsequence SEQ ID NO: 3 or a nucleic acid sequence called here forpractical reason SEQ ID NO X which, when aligned with SEQ ID N: 3, has:

a) a percentage of identical residues over SEQ ID

NO: 3 length of at least 50%, preferably of at least 70% and morepreferably of at least 90% and

b) a percentage of identical residues over said amino acid sequence SEQID NO X length of at least 80%, preferably of at least 90% and morepreferably of at least 95%,

or fragments thereof having at least 60 contiguous nucleotides.

According to the invention, the percentage of identical residues overSEQ ID NO: 3 length corresponds to the number of identical residuesbetween SEQ ID NO: X and SEQ ID NO: 3 divided by the number of residuesin SEQ ID NO: 3. When using GenomeQuest database, said percentage ofidentical residues over SEQ ID NO:3 length corresponds to querypercentage identity (% id Query), where query is SEQ ID NO:3.

According to the invention, the percentage of identical residues overSEQ ID NO: X length corresponds to the number of identical residuesbetween SEQ ID NO: X and SEQ ID NO: 3 divided by the number of residuesin SEQ ID NO: X. When using GenomeQuest database, said percentage ofidentical residues over SEQ ID NO: X length corresponds to subjectpercentage identity (% id Subject).

As used above, “fragments” means truncated sequences of SEQ ID NO:3,having at least 60 contiguous nucleotides, and coding for an activepeptide or polypeptide having an anti-angiogenic and anti-tumouractivity. In a particular embodiment, the fragments have the nucleicacid sequence of SEQ ID NO:13, SEQ ID:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, or SEQ ID NO:18. In another embodiment, the invention alsoencompasses nucleic acid fragments, which when aligned with fragments ofSEQ ID NO: 3 of at least 60 contiguous nucleotides and in particular SEQID NO: 13, 14, 15, 16, 17 or 18, have

a) a percentage of identical residues over the length of said fragmentsof SEQ ID NO: 3 of at least 50%, preferably of at least 70% and morepreferably of at least 90% and

b) a percentage of identical residues over said nucleic acid fragmentslength of at least 65%, preferably of at least 70% and more preferablyof at least 90%,

and encoding a peptide having an anti-angiogenic and anti-tumouractivity.

In another aspect, the invention relates to an expression vectorcomprising at least one nucleic acid sequence as defined above.

As used herein, “expression vector” means any plasmid or nucleic acidconstruct, which is used to introduce and express a specific nucleicacid sequence into a target cell.

The invention further relates, in a third aspect, to an activepolypeptide comprising the amino acid sequence of SEQ ID NO:4, or havingat least 50%, preferably 70%, more preferably 90% identity with theamino acid sequence of SEQ ID NO:4, or fragments thereof having at least20 contiguous amino acids, or peptides having at least 50%, preferably70%, more preferably 90% identity with the amino acid sequence of saidfragments, provided that said polypeptide is not SEQ ID NO:2, SEQ IDNO:22, or the polypeptide encoded by SEQ ID NO: 58 disclosed inUS2005/106644 or encoded by SEQ ID NO: 23.

SEQ ID NO: 22 corresponds to the amino acids sequence SEQ ID NO: 87disclosed in WO02/081731.

In a preferred embodiment, the invention relates to the activepolypeptide SEQ ID NO:4, or an amino acid sequence SEQ ID NO Y which,when aligned with SEQ ID N: 4, has:

a) a percentage of identical residues over SEQ ID NO: 4 length of atleast 50%, preferably of at least 70% and more preferably of at least90% and

b) a percentage of identical residues over said amino acid sequence SEQID NO Y length of at least 65%, preferably of at least 70% and morepreferably of at least 90%,

or fragments thereof having at least 20 contiguous amino acids.

As used herein, “peptide” means short molecules formed from the linking,in a defined order, of less than 100 amino acids.

As used herein, “polypeptide” means molecules formed from the linking,in a defined order, of at least 100 amino acids.

As used herein, “active polypeptide” means polypeptides which have abiological activity. In the present invention the polypeptides have ananti angiogenic and anti tumour activity.

According to the invention, the polypeptide as described above has ananti-tumour activity.

According to the invention, the percentage of identical residues overSEQ ID NO: 4 length corresponds to the number of identical residuesbetween SEQ ID NO: Y and SEQ ID NO: 4 divided by the number of residuesin SEQ ID NO: 4. When using GenomeQuest database, said percentage ofidentical residues over SEQ ID NO:4 length corresponds to querypercentage identity (% id Query), where query is SEQ ID NO:4.

According to the invention, the percentage of identical residues overSEQ ID NO: Y length corresponds to the number of identical residuesbetween SEQ ID NO: Y and SEQ ID NO: 4 divided by the number of residuesin SEQ ID NO: Y. When using GenomeQuest database, said percentage ofidentical residues over SEQ ID NO:Y length corresponds to subjectpercentage identity (% id Subject).

According to the invention, the fragments of SEQ ID NO:4 as describedabove correspond to truncated forms of SEQ ID NO:4, and have ananti-tumor activity. Said fragments preferably have an amino acidsequence of at least 20 contiguous amino acids of SEQ ID NO:4. In aparticular embodiment, the fragments have an amino acid sequence of atleast 37 contiguous amino acids. In another particular embodiment, saidfragments have the amino acid sequence of SEQ ID N:7 (90 amino acids),SEQ ID N:8 (77 amino acids), SEQ ID N:9 (66 amino acids), SEQ ID N:10(51 amino acids), SEQ ID N:11 (37 amino acids) or SEQ ID N:12 (20 aminoacids). In another embodiment, the invention also encompasses peptides,which when aligned with fragments of SEQ ID NO: 4 of at least 20contiguous amino acids and in particular SEQ ID NO: 7, 8, 9, 10, 11 or12, have

a) a percentage of identical residues over the length of said fragmentsof SEQ ID NO: 4 of at least 50%, preferably of at least 70% and morepreferably of at least 90% and

b) a percentage of identical residues over said peptide length of atleast 65%, preferably of at least 70% and more preferably of at least90%,

and having an anti-tumor activity.

In a particular embodiment, the active polypeptides according to theinvention are produced by the expression vector as defined above.

In a fourth aspect, the present invention relates to a medicamentcomprising at least one nucleic acid sequence, vector, or polypeptide asdescribed above.

In a fifth aspect, the invention relates to a pharmaceutical compositioncomprising at least one nucleic acid sequence, vector, or polypeptide asdescribed above, and one or more pharmaceutically acceptable excipients.

In a sixth aspect, the invention relates to a pharmaceutical compositioncomprising at least one nucleic acid sequence, vector, or polypeptide asdescribed above, and one or more pharmaceutically-acceptable excipients,for use in a method of treatment of cancer and/or tumors of the human oranimal body.

In a particular embodiment, the pharmaceutical compositions as describedabove further comprise at least one another active substance selectedfrom anti-angiogenic substances or anti-tumor substances. Thesesubstances may be chosen by the man in the art, regarding the effect tobe achieved. Preferably, these substances can be selected fromcisplatin, carboplatin, etoposide, ifosfamide, mitomycin, vinblastine,vinorelbine, gemcitabine, paclitaxel, docetaxel, and irinotecan, etc. .. . .

In a seventh aspect, the invention relates to a pharmaceuticalcomposition comprising effective amounts of

-   -   a polypeptide, a fragment, and/or a peptide as described above,        and    -   a platinum complex selected from the group consisting of        cisplatin and carboplatin.

Applicants surprisingly found that the combination of a polypeptideaccording to the invention with a platinum complex showed synergisticactivity.

By “synergistic”, it is meant, within the present invention, that thetotal effect of the combination of active principles is greater than theeffect of each active principle taken separately.

The medicament or composition useful in the practice of this inventionis administered to the mammal by known conventional routes. Themedicament or composition described herein may be administered by thesame route, or by different routes. For example, the medicament orcomposition may be administered to patients orally or parenterally(intravenously, subcutaneously, intramuscularly, intraspinally,intraperitoneally, and the like).

When administered parenterally, the composition is preferably formulatedin a unit dosage injectable form (solution, suspension, emulsion) withat least one pharmaceutically acceptable excipient. Such excipients aretypically nontoxic and non-therapeutic. Examples of such excipients arewater, aqueous vehicles such as saline, Ringer's solution, dextrosesolution, and Hank's solution and non-aqueous vehicles such as fixedoils (e.g., corn, cottonseed, peanut and sesame), ethyl oleate, andisopropyl myristate. Sterile saline is a preferred excipient. Theexcipient may contain minor amounts of additives such as substances thatenhance solubility, isotonicity, and chemical stability, e.g.,antioxidants, buffers, and preservatives. When administered orally (orrectally) the compounds will usually be formulated into a unit dosageform such as a table, capsule, suppository, or cachet. Such formulationstypically include a solid, semi-solid or liquid carrier or diluent.Exemplary diluents and excipients are lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineraloil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin,methylcellulose, polyoxyethylene, sorbitan monolaurate, methylhydroxybenzoate, propyl hydroxybenzoate, talc and magnesium stearate. Inpreferred embodiments, the pharmaceutical composition according to theinvention is administered intravenously.

According to the invention, the amount of polypeptide present in themedicament or composition is effective to treat susceptible tumors.Preferably, the polypeptide is present in an amount from 0.01 to 90% inweight, preferably from 0.1% to 10% in weight, more preferably from 1%to 5% in weight, in the medicament or in the composition. These amountsare routinely adaptable by the man in the art, who is able to choose thebest quantity to administer to a patient to achieve recovery.

In an eight aspect, the invention relates to the use of at least onenucleic acid sequence, vector, or polypeptide as described above, or ofthe medicament as described above, or of the pharmaceutical compositionas described above, for the treatment of cancers and/or tumors.

According to the invention, the tumors to be treated are preferablysolid tumors. More preferably, the tumors to be treated are selectedfrom sarcomas, carcinomas, and lymphomas. Examples of such tumors arebladder cancer, melanoma, breast cancer, non-Hodgkin's lymphoma, braincancer, bone cancer, colon and rectal cancer, liver cancer, pancreaticcancer, endometrial cancer, prostate cancer, kidney cancer, skin cancer(non-melanoma), thyroid cancer, lung cancer (small cell lung cancer andnon small cell lung cancer).

In a ninth aspect, the present invention relates to a method oftreatment comprising administering to a subject in need of treatment atleast one nucleic acid sequence, vector, or polypeptide as describedabove, or the medicament as described above, or the pharmaceuticalcomposition as described above, in an amount sufficient to inhibitcancer or tumor growth.

As used herein, “subject in need of treatment” means any human or warmblood animal who suffers from cancer or tumour.

In a particular embodiment, the invention relates to the method oftreatment as described above further comprising administering at leastone other anti-neoplastic or anti-tumor drug.

In these methods, administering comprises topical administration, oraladministration, intravenous administration, or intraperitonealadministration.

In an eight aspect, the present invention relates to a method oftreatment comprising administering to a subject in need of treatment aneffective amount of

-   -   a polypeptide, a fragment, and/or a peptide as described above,        and    -   a platinum complex selected from the group consisting of        cisplatin and carboplatin,        which is sufficient to inhibit cancer or tumor growth.

Applicants surprisingly found that the administration of both apolypeptide according to the invention and a platinum complex showedsynergistic effect.

In one embodiment, said polypeptide or fragments thereof and saidplatinum complex are administered simultaneously.

In another embodiment, said polypeptide or fragments thereof and saidplatinum complex are administered sequentially. Preferably, saidpolypeptide or fragments thereof and said platinum complex areadministered by separate routes, i.e. orally or parenterally(intravenously, subcutaneously, intramuscularly, intraspinally,intraperitoneally, and the like).

In a particular embodiment, said platinum complex is cisplatin.

In another particular embodiment said platinum complex is carboplatin.

The present invention will now be further described with reference tothe following non-limiting examples.

FIG. 1 is a diagram representing the % inhibition of endothelial cellproliferation in vitro with increasing concentrations of protein168A-T2.

FIGS. 2 a, 2 b, 2 c and 2 d are pictures of in vitro angiogenesis ofendothelial cells in different conditions:

FIG. 2 a: Control (Buffer Urea 2M)

FIG. 2 b: 168A-T2 3.5 μg/mL (0.2 μM)

FIG. 2 c: 168A-T2 6.9 μg/mL (0.4 μM)

FIG. 2 d: 168A-T2 13.6 μg/mL (0.8 μM)

FIGS. 3 a, 3 b, 3 c and 3 d are pictures of wound assay on endothelialcells performed in different conditions:

FIG. 3 a: Control (Buffer Urea 2M)

FIG. 3 b: 168A-T2 12 μg/mL (0.7 μM)

FIG. 3 c: 168A-T2 17 μg/mL (1 μM)

FIG. 3 d: 168A-T3 23 μg/mL (1.35 μM)

FIG. 4 is a diagram representing the % inhibition of kidney tumor cell(Biz) proliferation in vitro with increasing concentrations of protein168A-T2.

FIG. 5 is a diagram representing the % inhibition of lung tumor cell(Calu-6) proliferation in vitro with increasing concentrations ofprotein 168A-T2.

FIG. 6 is a graph representing Mean Tumor Volume (mm³) versus time(days) for different groups of mice treated according to example 8.

FIG. 7 is a graph representing Mean Relative Tumor Volume (without unit)versus time (days) for different groups of mice treated according toexample 8.

SEQ ID NO:1 corresponds to the nucleic acid sequence of gene 168.

SEQ ID NO:2 corresponds to the amino acid sequence of protein 168A.

SEQ ID NO:3 corresponds to the nucleic acid sequence of 168A-T2.

SEQ ID NO:4 corresponds to the amino acid sequence of 168A-T2.

SEQ ID NO:5 corresponds to the nucleic acid sequence of 168A-T2 withinvector pET30.

SEQ ID NO:6 corresponds to the amino acid sequence of 168A-T2 asproduced with the vector pET30.

SEQ ID NO:7 corresponds to the amino acid sequence of a fragment of168A-T2.

SEQ ID NO:8 corresponds to the amino acid sequence of a fragment of168A-T2.

SEQ ID NO:9 corresponds to the amino acid sequence of a fragment of168A-T2.

SEQ ID NO:10 corresponds to the amino acid sequence of a fragment of168A-T2.

SEQ ID NO:11 corresponds to the amino acid sequence of a fragment of168A-T2.

SEQ ID NO:12 corresponds to the amino acid sequence of a fragment of168A-T2.

SEQ ID NO:13 corresponds to the nucleic acid sequence coding for SEQ IDNO:7.

SEQ ID NO:14 corresponds to the nucleic acid sequence coding for SEQ IDNO:8.

SEQ ID NO:15 corresponds to the nucleic acid sequence coding for SEQ IDNO:9.

SEQ ID NO:16 corresponds to the nucleic acid sequence coding for SEQ IDNO:10.

SEQ ID NO:17 corresponds to the nucleic acid sequence coding for SEQ IDNO:11.

SEQ ID NO:18 corresponds to the nucleic acid sequence coding for SEQ IDNO:12.

SEQ ID NO:19 corresponds to the nucleic acid sequence of primer CDS5.

SEQ ID NO:20 corresponds to the nucleic acid sequence of primer CDS4.

EXAMPLE 1 Production of Protein 168A-T2 Synthesis of Insert 168A-T2:

First, gene 168A was cloned in pGEM®-T easy vector system (Promega®)according to known procedures (the vector obtained was called“pGEM-T-168A”).

Second, the insert T2 (SEQ ID NO:3), coding for the plasma membraneadjacent part of the extra-cellular domain of the protein 168A, wasamplified by PCR using the plasmid “pGEM-T-168A” and the two primersCDS5 (SEQ ID NO:19) and CDS4 (SEQ ID NO:20) (table 1).

TABLE 1 SEQ Primer ID NO Sequence 168A-cds-5 19GACGACGACAAGATGGCCTTTGATGTGTCCTG GTTTG 168A-cds-4 20GAGGAGAAGCCCGGTTCAGGGATACTTGAAGG CGTTCAGCACA

Third, the DNA sequence (SEQ ID NO:3) coding for the protein 168A-T2 wasinserted into the vector pET-30 EK/LIC (Novagen®) according to knownprocedures (pET-30-168A-T2). The nucleic acid sequence coding for168A-T2 within the pET-30 vector is given in SEQ ID NO:5.

The purified vector was then introduced in E. coli BL21(DE3)pLys forprotein production. Colonies were controlled for the presence of boththe vector end the insert by PCR.

The size of the produced protein 168A-T2 was 18 kD, which correspondedto the expected size, comprising the His-Tag at the N-terminal asconfirmed by sequencing. The amino acid sequence of the protein 168A-T2as produced is given in SEQ ID NO:6.

Extraction and Purification of the Protein 168A-T2

As the protein 168A-T2 was produced within the insoluble fraction of thebacteria, it necessitated an extraction in denaturating conditions

Following culture, bacteria were lyzed, centrifuged and the supernatantdiscarded. The insoluble fraction obtained was treated with Tris-HCl 20mM, urea 8 M, imidazol 5 mM, NaCl 0.5 M, GSH 5 mM, pH 8.0. After thistreatment, the suspension was centrifuged and the supernatant collected,filtered on 0.45 μm membranes to discard insoluble materials. Thefiltered extract was then used to purify the protein 168A-T2 by using aHis-Trap column (Amersham®) connected to a HPLC system (Amersham).

The purified protein obtained was diluted in 4 M urea and 0.3 Mimidazol. To remove these agents from the preparation, the solution wassubjected to dialysis at 4° C.

Following these steps of dialysis, the purified protein was centrifugedat 4,000×g for 15 min and filtered on 0.45 μm membranes to eliminatepossible precipitates. The purified protein preparation was controlledfor protein content according to the method described by Bradford in1976 (Anal. Biochem. 72:248-54) and by SDS-PAGE. The gels were analyzedusing the Gene Genius software to quantify the purity by image analysis.

To increase purity of the protein 168A-T2, we performed a secondpurification step by using ion exchange liquid chromatography. TheHisTrap purified preparation was diluted 3 times with the bufferTris-HCl 20 mM, pH 8, 2 M urea (to decrease the concentration of NaCl to50 mM), and loaded on MonoS column connected to a HPLC system run byUnicorn software (Amersham, GE, Saclay, France). The column was thenwashed extensively and eluted with a linear gradient of ionic force(0.05 M to 0.5 M NaCl in the Tris-HCl 20 mM buffer, pH 8, 2 M urea). Thepurified protein preparation was controlled for protein content both byBradford and by SDS-PAGE.

EXAMPLE 2 Test of Inhibition of Endothelial Cell Proliferation by168A-T2 In Vitro

HUVEC cells were cultured to confluency in complete EGM2-MV medium(Cambrex) at 37° C. and in 5% CO₂ humidified atmosphere. Cells were thencollected by trypsine-EDTA digestion (Versene, Eurobio). After 5 min,the enzymatic reaction was stopped by adding 3 ml of the culture mediumcontaining 5% FCS. Cells were then centrifuged at 220 g for 10 min atroom temperature, washed twice with 5 ml of culture medium, suspended incomplete culture medium, counted and adjusted to 50 000 cells/ml. Onehundred μL per well were then distributed to a 96-well cell culturegrade micro-plate (5 000 cells/well) and incubated with differentconcentrations of the purified protein 168A-T2 in Tris-HCl 20 mM buffer(pH 8), containing 150 mM NaCl and urea 2M; this buffer was used ascontrol.

After 42 hrs at 37° C., cell proliferation was measured using thiazolylblue tetrazolium bromide (MTT) method. Briefly, MTT (Sigma) wasdissolved in PBS at 5 mg/ml, the solution was filtered (0.22 μm) and 10μl were added to each well of the 96-well micro-plates. After 3 hrs ofincubation at 37° C., 5% CO₂ humidified atmosphere, the micro-plateswere centrifuged at 220×g for 10 min, the supernatant was discarded, andthe crystals dissolved by the addition of 100 μl of DMSO to each well.The optical density (OD) at 570 nm was then measured using μQuantmicro-plate reader (Bio-Tek Instrument gmbh, Colmar, France) coupled tothe KC4 (Bio-Tek) software. The OD was corrected by subtractingblank-well CD values (the OD values obtained from wells without cells),and the inhibition of cell proliferation was measured relative tocontrol (OD obtained from wells with untreated HUVEC representing themaximal proliferative response, i.e. 100%).

As shown in FIG. 1, protein 168A-T2 inhibited human endothelial cellproliferation in a dose dependent manner. This inhibition represented80% at 54 μg/mL (i.e. 3.1 μM) of protein 168A-T2.

EXAMPLE 3 Inhibition of In Vitro Angiogenesis by 168A-T2

The purified proteins 168A-T2 was tested in vitro on angiogenesis ofHUVEC induced by FGF2 and VEGF on Matrigel.

24 wells plates were prepared with 250 μL of BD Matrigel™/well and thenincubated 30 minutes in incubator. HUVEC cells were then prepared asdescribed in example 2 and 70 000 cells (in 0.5 mL) were seeded per welland incubated with different concentrations of the purified protein168A-T2, in Tris-HCl 20 mM buffer (pH 8), containing 150 mM NaCl andurea 2M; this buffer was used as control:

-   -   FIG. 2 a: Control (Buffer Urea 2M)    -   FIG. 2 b: 168A-T2 3.5 μg/mL (0.2 μM)    -   FIG. 2 c: 168A-T2 6.9 μg/mL (0.4 μM)    -   FIG. 2 d: 168A-T2 13.6 μg/mL (0.8 μM)

As shown in FIGS. 2 b, 2 c, and 2 d, protein 168A-T2 inhibited in vitroangiogenesis in a dose-dependent manner.

EXAMPLE 4 Inhibition of the Migration of Human Endothelial Cells by168A-T2

Cell migration was tested by the wound assay described by Sato andRifkin (J Cell Biol. 1988;107:1199) with few modifications. HUVEC grownin growth medium EGM-2MV (Cambrex) were seeded in 24-well plates at 80000 cells per well in 500 μL of growth medium and grown to confluence at37° C. in a humidified atmosphere containing 5% CO₂. Cells were scrappedwith a plastic tip on one line only. After wounding, the culture mediumwas changed for fresh medium (control, FIG. 3 a) or fresh mediumsupplemented with:

-   -   FIG. 3 b: 168A-T2 12 μg/mL (0.7 μM)    -   FIG. 3 c: 168A-T2 17 μg/mL (1 μM)    -   FIG. 3 d: 168A-T3 23 μg/mL (1.35 μM)

After 18 hours of culture, cells were observed and photographed underthe inverted microscope (Analysis, Olympus, Rungis, France).

As shown in FIGS. 3 b, 3 c and 3 d, protein 168A-T2 inhibited humanendothelial cells migration in a dose dependent manner.

EXAMPLE 5 Test of Inhibition of a Kidney Cancer Cell Line Proliferationby 168A-T2 In Vitro

BizX cell preparation of 50000 cells/mL was prepared in complete medium.In a 96 wells plate, 100 mL of the cell preparation was distributed ineach well and then incubated with different concentrations of 168A-T2(each concentration was tested in triplicate). After 48 hours ofincubation at 37° C., 10 mL of MTT (5 mg/L in water) were added in eachwell. After 3 hours of incubation at 37° C., the culture medium waseliminated and 100 mL of DMSO were added to solubilize MMT crystals. Theoptical density (OD) at 570 nm was then measured using μQuantmicro-plate reader (Bio-Tek Instrument gmbh, Colmar, France) coupled tothe KC4 (Bio-Tek) software. The OD was corrected by subtractingblank-well OD values (the OD values obtained from wells without cells),and the inhibition of cell proliferation was measured relative tocontrol (OD obtained from wells with untreated kidney tumor cellsrepresenting the maximal proliferative response, i.e. 100%).

As shown in FIG. 4, protein 168A-T2 inhibited up to 20% of proliferationof a kidney cancer line (BizX).

EXAMPLE 6 Test of Inhibition of a Lung Cancer Cell Line Proliferation by168A-T2 In Vitro

Calu6 cells were cultured and treated as described in example 5.

As shown in FIG. 5, protein 168A-T2 inhibited up to 40% of proliferationof a lung cancer line (Calu6).

EXAMPLE 7 Expression of Protein 168A in Tumor Samples

A number of different human tumor samples were screened for theexpression of the gene 168A. For each pathological sample, the peripheryof the tumor was separated from the core of the tumor, and theexpression of the gene 168A in these two area was compared after mRNAextraction followed by RT-PCR.

Kidney Tumor Samples

19 pathological biopsies from kidney tumors were analyzed. In 11 out of19 patients, the expression of 168A was much higher in the core than inthe periphery of the tumor. In 8 patients out of 19 patients, theexpression of 168A was much higher in the periphery of the tumor than inthe core.

Lung Tumor Samples

40 pathological biopsies from human lung tumors were analyzed. In 23 outof 40 patients, the expression of 168A was much higher in the core thanin the periphery of the tumor.

Colon Tumor Samples

33 pathological biopsies from human colon tumors were analyzed. In 13out of 33 patients, the expression of 168A was much higher in theperiphery than in the core of the tumor.

EXAMPLE 8 Test of 168A-T2 on Human Non-Small Cell Lung Cancer (CALU-6)Xenograft Model in Swiss Nude Mice In Vivo Preparation of CALU-6 Cells

CALU-6 cells were cultured as adherent cells in complete EMEM medium(Ref. CM1MEM18-01, batch No. 462502, Eurobio, France) 10% fetal calfserum (FCS; Ref. CVFSVF00-01, batch No. S13021, Eurobio, France) under a37° C., 5% CO₂ humidified atmosphere. They were amplified in 75cm²-flasks to reach 90×10⁶ cells.

At D0, CALU-6 cells (human lung carcinoma) were collected from 75cm²-flasks by removing the medium and adding 3 ml of trypsine-EDTA (Ref.CEZTDA00-0U, batch No. 633920, Eurobio, France). After 5 min ofincubation at 37° C., cells had detached from the plastic and theenzymatic reaction was stopped by adding 3 ml of EMEM medium containing10% fetal calf serum. Cells were then centrifuged at 700 g for 5 min atroom temperature. They were resuspended in serum-free EMEM culturemedium. Cells were counted and viability assessment by Trypan Blueexclusion (Ref. CSTCOL03-OU, batch No. 434511, Eurobio, France). Thenumber of viable CALU-6 cells was >99%. The number of cells was thenadjusted to 25×10⁶ cells/ml in serum-free medium.

Tumor Induction

Thirty healthy female Swiss Nude mice were anesthetized by IP injectionof Ketamine-Xylazine (80 mg/kg-12 mg/kg; Ref. K-113, Sigma, France).CALU-6 cells 5×10⁶ cells/mouse in 200 μl of serum-free medium) were thenimplanted subcutaneously in the right flank of each mouse. Mice wereobserved for 2 h post-implantation.

Treatment Schedule

At D12 post-implantation of the CALU-6 cells, the thirty mice wererandomized into four groups of 5 mice. Tumor volumes had reached 54 to296 mm³ and mean tumor volumes were not statistically different betweengroups after randomization.

The treatment schedule, starting D12 and ending D28, is summarized inTable 2.

-   -   Animals of group 1 were treated with the vehicle solution        (Tris-HCl pH 7.5, 2M Urea, 150 mM NaCl, 0.1 mM CaCl₂)    -   Animals of group 2 were treated with a solution of cisplatin in        physiological serum at a concentration of 0.5 mg/mL (CDDP,        cis-diamineplatinum(II) dichloride, Ref. P4394, batch No.        014K0993, Sigma, France, purity 100%, MW. 300),    -   Animals of group 3 were treated with the vehicle supplemented        with the test substance 168A-T2 at a dose of 15 mg/kg.    -   Animals of group 4 were treated with the vehicle supplemented        with the test substance 168A-T2 at a dose of 15 mg/kg, and        further received 5 mg/kg of CDDP.

Injections in groups 1, 2, 3 and 4 were performed according to theschedules Q2DX8, i.e. 1 quantity every two days, eight times.

Mice were observed for 2 hours post-injection. Ketamine/Xylazine (80mg/kg-12 mg/kg; Ref. K-113, Sigma, France) was used to anaesthetize theanimals before sacrifice by cervical dislocation. For all animals, thetumor size was measured twice a week with calipers. The tumor volume(mm³) was measured according to the formula: (length×width²)/2.

Statistical Studies

Data Outlined Below were Calculated:

-   -   Tumor growth curves were drawn using the mean tumor volumes        (MTV),    -   Mean Relative tumor volume (MRTV) was calculated as the ratio        between the MTV at time t and the volume at the time of        injection (t=D12),    -   Tumor growth inhibition (T/C, %) was evaluated as the ratio of        the median tumor volumes of treated groups versus vehicle group.

Statistical analyses of tumor volumes (TV), time to reach ‘TV’,tumor-doubling time (DT), relative tumor volume (RTV) and tumor growthinhibition (T/C) were performed for all groups. Data are expressed asmean±SD. Groups of data were normally distributed. Univariate analysiswere performed to assess differences between groups. Statisticalsignificance was then determined using the Student's t test. A P<0.05was considered as statistically significant. The Statistical analysiswas performed using XLSTAT (Addinsoft, France).

Body Weight

As shown in table 3, the vehicle had no impact: mouse behavior and bodyweight gain were normal and no animal died prematurely. No toxicity wasobserved during the course of the treatment with the test substance168A-T2 at the dose of 15 mg/kg, a slight body weight gain was observed(+2.45 g).

In contrast, an important toxicity was observed in groups 2, 4 treatedwith CDDP (−2.70 g and −2.35 g body weight loss respectively). Thedifference between group 1 versus 2 and 4 and group 3 versus 2 and 4 wasstatistically significant (p<0.0001) but the difference between group 2and 4 was not statistically significant.

TABLE 2 Animals Administration Treatment dose Administration TreatmentGroup n Treatment route (mg/kg/adm) volume schedule 1 5 Vehicle IP 0 10ml/kg Q2DX8 2 5 Cisplatin IP 5 Q2DX8 3 5 168A-T2 IP 15 Q2DX8 4 5 168A-T2& IP 15 Q2DX8 Cisplatin 5

TABLE 3 Mean body weight (MBW) of mice bearing CALU-6 tumors treatedwith the vehicle, CDDP at 5 mg/kg (schedule Q2DX8, G2), 168A-T2 at 10.0mg/kg (schedule Q2DX8, G3), combined 168A-T2 at 10.0 mg/kg and CDDP at 5mg/kg (schedule Q2DX8, G4) at D 12 and D 28. Treatment dose Group Testsubstance (mg/kg) MBW at D 12 (g) MBW at D 28 (g) MBWC D 12-D 28 (g) 1Vehicle 0 22.35 ± 1.17 24.40 ± 1.19 +2.36 (±0.59) 2 Cisplatin 5.00 21.40± 0.85 18.69 ± 1.97 −2.70 (±1.57) 3 168A-T2 15.0 21.74 ± 0.89 24.20 ±1.37 +2.45 (±0.65) 4 168A-T2 + Cisplatin 15.0 + 5.00 20.74 ± 1.41 18.39± 0.74 −2.35 (±1.99)

The results of mean tumor volume (MTV), mean relative tumor volume(MRTV) and tumor growth parameters (T/C) are shown in FIGS. 6, 7 and inTables 4, 5 and 6.

The MTV (Table 4, FIG. 6) was decreased at D28 in mice of group 2treated with CDDP (742.44 mm³) compared to mice of the vehicle group 1(1233.44 mm³). The MTV at D28 was also decreased in group 3 treated withthe test substance 168A-T2 at 15 mg/kg with 1 injection per two days(615.96 mm³). The most important MTV decrease was obtained for animalsof group 4 (317.17 mm³). The difference between group 1 and the 2 groupstreated with the test substance reach the statistical significativity(p<0.0001 vs 4−p=0.003 vs 3). The difference between group 2 and 4 wasalso significant (p=0.001). In contrast no statistical difference wasobserved between group 2 (CDDP alone) and group 3 (168A-T2 alone).

TABLE 4 Mean tumor volume (MTV) of animals bearing CALU-6 cells andtreated with vehicle (group 1), CDDP alone (Group 2), 168A-T2 (10.0mg/kg) (Group 3), or combined with 168A-T2 and CDDP (Group 4) accordingto the scheduled treatment Q2DX8. MTV (mm³) Group D 12 D 14 D 16 D 18 D20 D 22 D 24 D 26 D 28 1 142.61 252.49 365.52 522.27 784.61 588.53803.89 1044.02 1233.44 2 142.60 351.15 291.29 329.18 386.84 470.59595.00 527.98 742.44 3 159.22 181.94 264.20 322.99 391.90 485.42 642.24524.95 615.96 4 137.68 157.35 250.14 226.39 290.70 311.24 387.85 334.10317.17

These results were confirmed by the analysis of the MRTV (table 5, FIG.7). The MRTV for animals of group 1 was 8.24 at D28. For animals ofgroup 2, i.e. treated with CDDP, the MRTV at D28 was 5.98; for animalsof group 3, i.e. treated with 168A-T2 (15 mg/kg), the MRTV at D28 was4.51; eventually, for animals of group 4, i.e. treated with both CDDPand 168A-T2, the MRTV was 2.18.

TABLE 5 Mean Relative tumor volume (MRTV) of animals bearing CALU-6cells and treated with vehicle (group 1), CDDP alone (Group 2), 168A-T2(10.0 mg/kg) (Group 3), or combined with 168A-T2 and CDDP (Group 4)according to the scheduled treatment Q2DX8. MRTV Group D 12 D 14 D 16 D18 D 20 D 22 D 24 D 26 D 28 1 1 1.83 3.17 2.51 3.68 4.16 5.68 7.49 8.242 1 3.11 2.39 2.66 3.08 3.70 4.84 4.11 5.98 3 1 1.15 1.93 2.08 2.60 3.474.83 4.14 4.51 4 1 1.09 1.80 1.70 2.10 2.28 2.72 2.33 2.18

As shown in table 5 and FIG. 7, MRTV reached 2.18 at D28 for the animalsof group 4, which confirmed the synergistic efficacy of 168A-T2 withCisplatin. Moreover, cisplatin used alone (group 2) or 168A-T2 usedalone (group 3) showed close MRTV at D28, suggesting that 168A-T2 isalso a potent mono-therapy anti-tumor agent.

TABLE 6 Growth inhibition based on T/C ratio T/C ratio (%) Day D 14 D 16D 18 D 20 D 22 D 24 D 26 D 28 G2 −70%   24% −6% 17% 11% 15% 45% 27% G337% 39% 17% 29% 16% 15% 45% 45% G4 40% 43% 32% 43% 45% 52% 69% 74%

The T/C ratio (table 6), which is a parameter of tumor growthinhibition, revealed a slight anti-tumoral activity of the testsubstance when used as a monotherapy as it reduced by 27% tumor sizecompared to the vehicle-treated group 1. However, when combined withCDDP, the inhibition rate reached 74% reduction of tumor size relativeto the vehicle-treated group 1.

These results directly demonstrate that 168A-T2 has a potentanti-tumoral activity when it is used alone or in combination with acytotoxic agent such as CDDP.

1. A nucleic acid sequence SEQ ID NO:3, or a nucleic acid sequence SEQID NO X which, when aligned with SEQ ID N: 3, has: a) a percentage ofidentical residues over SEQ ID NO: 3 length of at least 50%, and b) apercentage of identical residues over said nucleic acid sequence SEQ IDNO X length of at least 80%, or fragments thereof having at least 60contiguous nucleotides.
 2. An expression vector comprising at least onenucleic acid sequence according to claim
 1. 3. An active polypeptide SEQID NO:4, or an amino acid sequence SEQ ID NO Y which, when aligned withSEQ ID N: 4, has: a) a percentage of identical residues over SEQ ID NO:4 length of at least 50%, and b) a percentage of identical residues oversaid amino acid sequence SEQ ID NO Y length of at least 65% or fragmentsthereof having at least 20 contiguous amino acids.
 4. An activepolypeptide according to claim 3, wherein said active polypeptide isproduced by an expression vector comprising at least one nucleic acidsequence SEQ ID NO:3, or a nucleic acid sequence SEQ ID NO X which, whenaligned with SEQ ID NO: 3, has: a) a percentage of identical residuesover SEQ ID NO: 3 length of at least 50%, and b) a percentage ofidentical residues over said nucleic acid sequence SEQ ID NO X length ofat least 80%, or fragments thereof having at least 60 contiguousnucleotides.
 5. The polypeptide according to claim 3, wherein saidpolypeptide has an anti-angiogenic and an anti-tumour activity.
 6. Amedicament comprising a component selected from the group consisting ofat least one nucleic acid sequence according to claim 1, at least oneexpression vector comprising said nucleic acid sequence, and at leastone polypeptide SEQ ID NO:4, or an amino acid sequence SEQ ID NO Ywhich, when aligned with SEQ ID N: 4, has: a) a percentage of identicalresidues over SEQ ID NO: 4 length of at least 50%, and b) a percentageof identical residues over said amino acid sequence SEQ ID NO Y lengthof at least 65% or fragments thereof having at least 20 contiguous aminoacids.
 7. A pharmaceutical composition comprising a component selectedfrom the group consisting of at least one nucleic acid sequenceaccording to claim 1, at least one expression vector comprising saidnucleic acid sequence, and at least one polypeptide SEQ ID NO:4, or anamino acid sequence SEQ ID NO Y which, when aligned with SEQ ID N: 4,has: a) a percentage of identical residues over SEQ ID NO: 4 length ofat least 50%, and b) a percentage of identical residues over said aminoacid sequence SEQ ID NO Y length of at least 65% or fragments thereofhaving at least 20 contiguous amino acids; and one or morepharmaceutically acceptable excipients.
 8. A pharmaceutical compositioncomprising a component selected from the group consisting of at leastone nucleic acid sequence according to claim 1, at least one expressionvector comprising said nucleic acid sequence, and at least onepolypeptide SEQ ID NO:4, or an amino acid sequence SEQ ID NO Y which,when aligned with SEQ ID N: 4, has: a) a percentage of identicalresidues over SEQ ID NO: 4 length of at least 50%, and b) a percentageof identical residues over said amino acid sequence SEQ ID NO Y lengthof at least 65% or fragments thereof having at least 20 contiguous aminoacids; and one or more pharmaceutically acceptable excipients, for usein a method of treatment of cancer and/or tumours of the human or animalbody.
 9. A pharmaceutical composition according to claim 7, furthercomprising at least one another active substance selected fromanti-angiogenic substances or anti-tumour substances.
 10. Apharmaceutical composition comprising effective amounts of a polypeptideaccording to claim 3, and a platinum complex selected from the groupconsisting of cisplatin and carboplatin.
 11. The pharmaceuticalcomposition according to claim 7, being in a form suitable for topical,systemic, oral, subcutaneous, transderm, intramuscular orintra-peritoneal administration.
 12. The pharmaceutical compositionaccording to claim 10, wherein said polypeptide is present in an amountfrom 0.01 to 90% in weight.
 13. A method for the treatment of cancersand/or tumours, comprising administering to a subject in need thereof aneffective amount of a component selected from the group consisting of anucleic acid sequence according to claim 1, an expression vectorcomprising said nucleic acid sequence, and a polypeptide according toSEQ ID NO:4, or an amino acid sequence SEQ ID NO Y which, when alignedwith SEQ ID N: 4, has: a) a percentage of identical residues over SEQ IDNO: 4 length of at least 50%, and b) a percentage of identical residuesover said amino acid sequence SEQ ID NO Y length of at least 65% orfragments thereof having at least 20 contiguous amino acids.
 14. Themethod according to claim 13, wherein the tumours are solid tumours. 15.The method according to claim 13, wherein the solid tumours are selectedfrom sarcomas, carcinomas, and lymphomas.
 16. A method for the treatmentof cancers and/or tumours, comprising administering to a subject in needthereof an effective amount of the pharmaceutical composition accordingto claim
 7. 17. A method for the treatment of cancers and/or tumours,comprising administering to a subject in need thereof an effectiveamount of the pharmaceutical composition according to claim
 9. 18. Amethod for the treatment of cancers and/or tumours, comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition according to claim
 10. 19. The pharmaceuticalcomposition according to claim 12, wherein said polypeptide is presentin an amount from 0.1% to 10% in weight.
 20. The pharmaceuticalcomposition according to claim 19, wherein said polypeptide is presentin an amount from 1% to 5% in weight.